Evaluation of commercially available antibodies and fluorescent conotoxins for the detection of surface ganglionic acetylcholine receptor on the neuroblastoma cell line, IMR-32 by flow cytometry.
J Immunol Methods
; 498: 113124, 2021 11.
Article
em En
| MEDLINE
| ID: mdl-34425081
ABSTRACT
Commercially available antibodies that bind to the human muscle acetylcholine receptor (ACHR) have been validated previously for flow cytometric use (Keefe et al., 2009; Leite et al., 2008; Lozier et al., 2015). Despite a multitude of commercially available antibodies to other nicotinic ACHRs, validation in a wide variety of immunoassay formats is lacking; when studied, a large proportion of these antibodies have been deemed not fit for most research purposes (Garg and Loring, 2017). We have recently described a flow cytometric immunomodulation assay for the diagnosis of Autoimmune Autonomic Ganglionopathy (AAG) (Urriola et al., 2021) that utilises the monoclonal antibody mab35(Urriola et al., 2021) which is specific for ganglionic ACHR (gnACHR) that contain α3 subunits (Vernino et al., 1998). Other fluorescent ligands for α3-gnACHR have not been validated for flow cytometric use. We investigated 7 commercially sourced antibodies and 3 synthetic fluorescent novel conotoxins purported to specifically bind to the extracellular domains of the gnACHR, and compared the results to staining by mab35, using flow cytometry with the neuroblastoma cell line IMR-32. We also evaluated the degree of non-specific binding by depleting the cell membrane of the relevant acetylcholine receptor with a pre-incubation step involving the serum from a patient with Autoimmune Autonomic Ganglionopathy containing pathogenic antibodies to the ganglionic acetylcholine receptor. None of the assessed conotoxins, and only one antibody (mab35) was found to perform adequately in flow cytometric staining of the native ganglionic acetylcholine receptor.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Doenças do Sistema Nervoso Autônomo
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Receptores Colinérgicos
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Doenças Autoimunes do Sistema Nervoso
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Conotoxinas
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Citometria de Fluxo
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Corantes Fluorescentes
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Gânglios Autônomos
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Anticorpos Monoclonais
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Neuroblastoma
Tipo de estudo:
Diagnostic_studies
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Evaluation_studies
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Prognostic_studies
Limite:
Humans
Idioma:
En
Revista:
J Immunol Methods
Ano de publicação:
2021
Tipo de documento:
Article